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(Chapter 14) Mammalian Artificial Chromosomes and Clinical Applications for Genetic Modification of Stem Cells - An Overview

(Chapter 14) Mammalian Artificial Chromosomes and Clinical Applications for Genetic Modification of Stem Cells - An Overview

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Gyula Hadlaczky (ed.),
Mammalian Chromosome Engineering: Methods and Protocols
, Methods in Molecular Biology, vol. 738,DOI 10.1007/978-1-61779-099-7_14, © Springer Science+Business Media, LLC 2011
Chapter 14
Mammalian Artifcial Chromosomes and Clinical Applications or Genetic Modifcation o Stem Cells: An Overview
Robert L. Katona, Sandra L. Vanderbyl, and Carl F. Perez
Modiying multipotent, sel-renewing human stem cells with mammalian articial chromosomes (MACs),present a promising clinical strategy or numerous diseases, especially ex vivo cell therapies that can ben-et rom constitutive or overexpression o therapeutic gene(s). MACs are nonintegrating, autonomously replicating, with the capacity to carry large cDNA or genomic sequences, which in turn enable potentially prolonged, sae, and regulated therapeutic transgene expression, and render MACs as attractive genetic vectors or “gene replacement” or or controlling dierentiation pathways in progenitor cells. The
status quo 
is that the most versatile target cell would be one that was pluripotent and sel-renewing to addressmultiple disease target cell types, thus making multilineage stem cells, such as adult derived early progeni-tor cells and embryonic stem cells, as attractive universal host cells. We will describe the progress o MACtechnologies, the subsequent modications o stem cells, and discuss the establishment o MAC platormstem cell lines to acilitate proo-o-principle studies and preclinical development.
Key words:
Mammalian articial chromosomes, MACs, Embryonic stem cells, Adult stem cells,Gene therapy, Transgenic animals
 Various groups worldwide have developed mammalian articialchromosomes (MACs) and these technologies have been exten-sively reviewed (1–18). The procedures to generate MACs allinto three broad categories: (1) the de novo ormation by cen-tromere seeding (“bottom-up” approach) (19–30), (2) the trun- cation o normal chromosomes or the modication o naturally occurring minichromosomes (“top-down” approach) (31–42),and (3) the induction o the intrinsic large-scale amplication o 
1. Introduction
1.1. Background on Mammalian Artifcial Chromosomes 
200Katona, Vanderbyl, and Perez
pericentromeric heterochromatin to generate satellite DNA-basedarticial chromosomes (SATACs and ACEs) (43–49). All three processes generate MACs with unctioning centromeres tomaintain nuclear location and to participate in mitotic/meioticsegregation; telomeres to preserve integrity and autonomy (e.g.,nonintegrating and nontranslocating); and origins o DNA repli-cation to duplicate genetic inormation required or transmissionto daughter cells. While various transgenes have been incorporated into cen-tromere-seeded MACs (50, 51), they have restricted clinical appli-cations due to the necessity o de novo chromosome ormation.Furthermore, in all reported cases, the newly ormed MACs arecomposed o signicantly more DNA than the original transectedcomponents, strongly suggesting that endogenous and unknownDNA had been incorporated. This issue raises saety questions which must be resolved beore considering this technology orgene therapy. Nevertheless, this methodology has been valuable orthe study o the structure and unction o centromeres (52–54).For MACs to unction as genetic vectors, they should(1) possess large DNA payload capacity o 1 Mb or greater, (2) acili-tate ecient gene(s) loading, (3) be capable o being transerredrom one cell to another, and (4) enable stable, high-level trans-gene expression. The two MAC technologies that have made themost progress in addressing these stringent requirements are aclass o truncated natural chromosomes (21
qHACs and21
pqHACs) and satellite DNA-based articial chromosomes(SATACs and ACEs).
qHACs and 21
. By systematically deletingeuchromatin rom natural chromosomes, truncation-derivedMACs preserve unctioning centromeres, telomeres, and DNA origins o replication, while minimizing potential gene dosageeects in host cells. O particular interests are the two MACs that were generated by telomere-directed truncation o the distal q(“long”) arm o human acrocentric chromosome 21 generating21
qHAC, and the subsequent truncation o the remaining dis-tal p (“short”) arm generating 21
pqHAC (42,55). O note, the remaining p arm o 21
qHAC is assumed to encode only anrDNA gene tandem array and pericentromeric heterochromatin,and hence should be genetically “neutral.” A single loxP targetedintegration site has been inserted into both
HACs, enablingthe “loading” o various single transgene sequences by Cre-recombinase mediated DNA recombination. However, serialmultiple loadings are hindered by the competing integration andexcision processes that are inherent in the cre–loxP system, whichincreases the probability that the original integrated transgene would excise during the introduction o additional constructs.Thereore to attain greater transgene copy number, the singleplasmid targeting vector must encode multiple transgenes, limiting
201Mammalian Artifcial Chromosomes and Clinical Applications
the versatility o this recombination system. Transgenes that havebeen eciently loaded and expressed rom these vectors includehypoxanthine guanine phosphoribosyl transerase (HPRT), eryth-ropoietin (EPO), enhanced green fuorescent protein (EGFP),antibody/cytokine receptor chimera, DNA-dependent proteinkinase catalytic subunit (DNA-PKcs, critical or DNA repair),proinsulin, human dystrophin, CD40L, the human P53, andtelomerase (42,55–64).
. SATACs and ACEs primarily consist o heterochroma-tin and pericentromeric sequences, which are purported to bedevoid o gene-coding sequences beyond rDNA (13,43–48). There is no evidence in humans that amplication o these peri-centromeric sequences is deleterious to an individual, as polymor-phisms in the short arms o acrocentric chromosomes have beenshown to consist o amplied pericentromeric heterochromatinand/or rDNA (65–73) and have been inherited with no adverseeects (74–78). Furthermore, supernumerary chromosomesderived rom amplied pericentromeric heterochromatin and/orrDNA sequences o acrocentric chromosomes have been shownto be stably inherited and through 1–3 generations in humans without any deleterious eects o phenotypes (79–85).  Additionally, ACEs have been engineered with multiple (>50)site-specic recombination acceptor sites (
P), which enable theunidirectional loading o heterologous DNA (encoding an
Brecombination donor site) via a mutant lambda integrase (48, 49).The DNA recombination is unidirectional in the context o themammalian cells, given that the engineered integrase lacks thebacterial co-actors necessary or excision. The combination o the multiple
P sites on the ACE and the unidirectional mutantintegrase enables multiple loadings (during a single transection)or sequential loadings (via multiple transections) o transgenes(48,86). Transgenes that have been eciently loaded and expressed rom ACEs include EGFP, red fuorescent protein(RFP), EPO, monoclonal antibodies (Mabs), and therapeuticgalactocerebrosidase transgenes to treat Krabbe disease in theTwitcher mice (48, 49,86–89). In addition, specic MAb pro- ductivities o 55 pg/cell/day and 4 g/L yields in nonoptimizedbioreactors have been attained ater transerring MAb-ACEs to various industrial strains o CHO cells, demonstrating high level ACE-encoded transgene expression (87).The delivery o transgene-loaded MACs to primary mammaliancells is a undamental challenge or gene therapy applications. While microcell-mediated chromosome transer (MMCT) has
2. Methods
2.1. Transer o MACs 

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